The goal of this project is to characterize the shape, molecular weight distribution and elemental composition of individual macromolecules and macromolecular assemblies. Such assemblies are critical to many cell functions, and their behavior in vitro reflects their function and regulation in intact cells. This project depends on a unique instrument a low~temperature, high~resolution, field~emission scanning transmission electron microscope (STEM) for molecular weight mapping and chemical analysis by parallel electron energy loss spectroscopy (PEELS) of directly frozen thin films and ultrathin cryo~sections of directly frozen tissues. Dark~field molecular weight mapping of squid brain kinesin has revealed a new conformation of this motor protein, in which the kinesin light chain end is folded back onto the stalk of the molecule near its hinge; this results in an apparently shortened stalk region. The potential significance of this conformation is suggested by mass analysis of kinesin~microtubule complexes, where we have found that single kinesins can crossbridge microtubule pairs with a typical spacing of 25 nm, consistent only with the shortened conformation of kinesin. The ability of single kinesins to crossbridge microtubules implies a second, previously unrecognized, microtubule binding site on the light~chain end of kinesin, and suggests that kinesin may play a role in stabilization of micro~tubule arrays and in microtubule sliding. We have applied a new method based on analyzing the valence electron region of a low~dose PEELS map of frozen~hydrated sections to determine the optimal thickness of cryosections for PEELS elemental analysis and to measure the distribution of water within Purkinje cell dendrites. This method in combination with high~dose PEELS spectrum imaging has been used to measure calcium in Purkinje dendrites with a fourfold improvement in sensitivity.